Human Allergen Specific IgE ELISA Assay (Product Insert)

Principle of Procedure:

                   Solid phase capture sandwich ELISA assay using a microwell format.

Shelf Life:

                  The expiration date for the package and each component is stated on the label(s). Store all components at 2°- 8° C. 

                  Do not freeze all or in part.

Materials Supplied:

Allergen coated microwell strips 12 x 8 with plastic frame

HRP conjugated anti-human IgE   - 1 X 12mL

TMB/peroxide substrate color developer II - 30ml

               Sulfuric acid termination reagent (0.5N) - 12mL

                   15 X Wash buffer concentrate - 60mL

Limitations of the Procedure:

                  No single assay should be used as the only basis for arriving at a diagnostic conclusion.
              For invitro diagnostic use.

Dynamic Range:

                   0.35 - 0.70  PRU/mL, depending on the type of allergen used.

Reproducibility:

                   C.V. 6% - 10%

Assay Procedure:

*Caution: All human fluids should be treated as infectious agents that could carry HIV.

For each specimen, place one strip of allergen coated microwells in the plastic frame and label the corresponding schematic with the patient’s names or ID numbers.

1.      Bring all reagents to 25ºC.

2.      Add 100 uL of serum or plasma to each microwell.

3.      Incubate for 2 hours at 25ºC but not greater than 30ºC.

4.      Decant and wash five times with previously diluted wash buffer (see "preparation of reagents.") Firmly grip and pound upside down on folded, clean dry paper towels after the last decantation.

5.      Add 100uL of HRP conjugated anti=human IgE to each well respectively.

6.      Incubate for 2 hours 25ºC but not greater than 30ºC.

7.      Decant and wash as in "step 5".

8.      Add 100uL of TMB/peroxide substrate (Colorburst TM Blue II) to each well respectively.

9.   Incubate for 30 minutes at 25ºC but not greater than 30ºC.

10.   Add 100uL of termination reagent (0.5N sulfuric acid) to each well respectively.

11.   Read the optical density (O.D.) at 450 nm using a standard microwell plate reader zeroed on the negative control well.

O.D. at 450nm                        Class:

0.000-0.250                                      Class 0 (Negative)

0.250-0.350                                       Class I  (Equivocal)

0.350-0.450                                       Class II (True Positive)

0.450-0.550                                       Class III

>0.550                                               Class IV

Preparation of Reagents:

Wash Buffer:

Prior to performing the assay, dilute 1 part of the 15X Wash Buffer concentrate in 14 parts of reagent grade water. Mix thoroughly and dispense into a squeeze wash bottle as needed.